Nitric oxide (NO) is implicated in the pathogenesis of migraine and as a target for therapy. Inhibition of NO synthase ameliorates migraine headaches, and typical headaches develop in migraineurs; (but not normals) with a 4-6 hr latency after nitroglycerin (GTN) infusion. Our preliminary data in a rodent model was directed toward explaining the unusually long headache latency following a 30 min infusion of relatively short-lived molecules (NO, GTN). Our data indicate that both GTN and sodium nitroprusside infusion promote gene up-regulation and a cascade of signaling events within pain-generating meningeal tissues (dura mater), characterized by increased iNOS, IkB-alpha mRNA levels, NFkB translocation, IL-1 beta production, and up-regulation of genes associated with cell stress (Heat-shock protein-27, metallothioneins 1 and 2, Tis-21, Cu/Zn superoxide dismutase). Importantly, changes in meningeal iNOS protein expression follow a time course consistent with GTN-induced migraine in humans, thereby suggesting that NO signaling and/or NO-related oxidative stress promote cytokine induction and a meningeal inflammatory response (e.g. protein leakage, as shown in preliminary experiments) relevant to migraine in susceptible individuals. Based on preliminary findings, we propose 5 aims to test the hypothesis that GTN infusion leads to oxidative/nitrergic stress and that iNOS expression in a subpopulation of dural cells, followed by activation/sensitization of trigemino-vascular neurons. Aim 1 will expand the characterization to determine the dose and time-dependency of the iNOS response to GTN infusion. Experiments will also determine the cell source of iNOS and characterize the constellation of mononuclear cells using selective antibodies. Aim 2 will examine upstream mechanisms of iNOS induction, to test the hypothesis that oxidative stress and/or nitrergic mechanisms contribute to the increased iNOS expression after GTN infusion. Aim 3 will dissect the cascade of transcriptional regulation of iNOS in meningeal tissues to identify relevant therapeutic targets. Aim 4 (in collaboration with Dr. R. Burstein) will examine the extent to which iNOS expression and meningeal cytokine up-regulation following GTN promotes increased basal firing of trigeminal brainstem nuclear complex (TBNC) neurons or augmented responses to meningeal stimulation (sensitization). Aim 5 will examine the functional significance of iNOS induction (COX-2 expression and protein leakage that can be blocked by NOS inhibition and/or by administering 5-HT1B/D receptor-selective acute anti-migraine drugs). By so doing, we aim to define the biological underpinnings of migraine headache and to identify therapeutic targets by exploring NO as potential headache generator in meningeal tissues.